Self-immolative probes for enzyme activity detection

ABSTRACT

Provided is a compound having the structure:
 
(SIG)-(SI-MOD) m  
 
where SIG is a signaling molecule, SI is a self-immolative structure bound to SIG such that SIG has a reduced signal relative to the signal of SIG without SI, MOD is a moiety bound to SI that is subject to modification by an activator, and m is an integer from 1 to about 10. When MOD is modified by an activator, SI is destabilized and self-cleaved from SIG such that SIG generates an increased signal. Also provided are methods of determining whether a sample, such as a cell, comprises an activator, such as a nitroreducase, using the compound. Further provided are methods of determining whether a mammalian cell is hypoxic using the compound where nitroreductase is the activator. A method of detecting a microorganism that comprises a nitroreductase using the compound where nitroreductase is the activator is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/416,119 filed Jan. 26, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/447,817 filed Jul. 31, 2014 (now U.S. Pat. No.9,574,222), which is a divisional of U.S. patent application Ser. No.12/927,497 filed Nov. 16, 2010 (now U.S. Pat. No. 8,828,678), thecontents of all of which are incorporated by reference herein in theirentirety.

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present invention generally relates to the field of reagents forvisual detection, quantification and localization of cells. Morespecifically, probes are provided that increase their signal uponexposure to specific enzyme or chemical analyte presence.

(2) Description of the Related Art

Signaling molecules that are responsive to the intracellular environmentare indispensable tools for fast and accurate detection and measurementof both physiological and pathological processes. However, only alimited number of rationally designed probes or “signalophores” capableof detecting intracellular organic biomolecules exist.

For a chemical molecule to be a suitable signalophore, it should meetseveral conditions. First, it should have favorable spectral properties,and be detectable by readily available sources and filter systems.Second, it should exhibit a significant signal enhancement triggered bythe presence of a specific enzyme activity or analyte to be detected.For the signal-to-background ratio, and as a result the sensitivity ofthe probe to be maximized, the probe should preferably be“signalogenic”—in a “no-signal” form in the absence of the enzyme oranalyte and in “signal on” form in their presence. One of the types ofprobes fulfilling the above criterion are self-immolative probes.

The concept of self-immolative substrates has been successfully used indesigning several prodrugs where the active drug is released upon theactivation by a specific chemical trigger. See, e.g., U.S. Pat. Nos.7,754,681 and 7,445,891. That approach was also used, albeit to muchlesser extent, for making markers for probing and detecting specificbiological processes and phenomena.

U.S. Pat. No. 7,534,902 describes fluorogenic assays based on the use ofa group of self-immolative markers containing a so-called trimethyllock, which is an aromatic self-immolative group that comprises threemethyl groups. Trimethyl locks have been used for detection andmeasurement of several enzymatic activities including esterases(Chandran et al., 2005; Lavis et al., 2006), DT diaphorase (DTD) (Huanget al., 2006), and cytochrome P450 (Yatzeck et al., 2008).

Self-immolative dendrimers that release multiple fluorescent moietiesupon activation have also been developed (US Patent PublicationUS2005/0271615; Danieli and Shabat, 2007).

Several latent probes have also been designed utilizing a benzylcarbamate self-immolative moiety. A fluorescent image contrast agentselectively activated by prostate specific antigen was described byJones et al. (2006), while Pires et al. (2008) evaluated cellularglutathione fluorescence imaging using a latent rhodamine derivative.

Substituted benzyl groups as self-immolative substrates were also usedby Nakata et al. (2009) for preparing bioreductively-activatedfluorescent pH probes for tumor hypoxia imaging. Richard et al. appliedthat group for preparing a chemiluminescent probe for in vitro detectionof protease activity while a long-wavelength latent fluorogenicsubstrate was utilized as an indicator for dehydrogenase-coupledbiosensors (Huang et al., 2010).

Several other self-immolative groups have been used for determination ofenzyme activities as well. A traceless linker that is stable underphysiological conditions but spontaneously decomposes to ahemithioaminal intermediate upon protease activation is taught by Meyeret al. (2008). Additionally, a Waldmann traceless linker has beenutilized for peptidase probes (Richard et al., 2008a). A penicillin Gacylase fluorogenic probe is also described by Richard et al., 2008b.Further, a self-immolative disulfide linker carboxylic acid was used toprepare biotin-containing fluorogenic probes for internalization anddrug release (Ojima et al., 2008). See also Sagi et al., 2008.Additional self-immolative substrates for detecting enzymes aredescribed in Gao et al. (2003), Duimstra et al. (2005), and Ho et al.(2006).

There is a need for further self-immolative signalogenic markers with ahigh signal-to-background ratio that are sensitive to specific enzyme oranalyte triggers. The present invention addresses that need.

SUMMARY OF THE INVENTION

The present invention provides several self-immolative probes that areuseful for detecting enzymes or other activators.

In some embodiments, a compound is provided that comprises thestructure:(SIG)-(SI-MOD)_(m)

wherein SIG is a signaling molecule, SI is a self-immolative structurebound to SIG such that SIG has a reduced signal relative to the signalof SIG without SI, MOD is a moiety bound to SI that is subject tomodification by an activator, and m is an integer from 1 to about 10. Inthese embodiments, when MOD is modified by an activator, SI isdestabilized and self-cleaved from SIG such that SIG generates anincreased signal.

In other embodiments, a method of determining whether a sample comprisesan activator is provided. The method comprises (a) incubating the samplewith the above-identified compound for a time and under conditionssufficient for MOD to be modified by the activator; and (b) determiningwhether SIG generates a greater signal than the signal generated by thecompound without the activator. In these embodiments, a greater signalindicates that the sample comprises the activator.

Also provided is a method of determining whether a cell comprises anitroreductase. The method comprises (a) incubating the cell with theabove-identified compound, where nitroreductase is the activator, for atime and under conditions sufficient for the compound to enter the celland be exposed to a nitroreductase if present in the cell; and (b)determining whether SIG generates a greater signal than the signalgenerated by the compound when not exposed to a nitroreductase. In theseembodiments, a greater signal indicates that the cell comprises thenitroreductase.

Additionally provided is a method of determining whether a mammaliancell is hypoxic. The method comprises (a) incubating the cell with theabove-identified compound, where nitroreductase is the activator, for atime and under conditions sufficient for the compound to enter the celland be exposed to a nitroreductase if present in the cell, wherein thenitroreductase is indicative or hypoxia in the cell; and (b) determiningwhether SIG generates a greater signal than the signal generated by thecompound when not exposed to a nitroreductase. In this method, a greatersignal indicates that the cell is hypoxic.

A method of detecting a microorganism that comprises a nitroreductase isalso provided. The method comprises (a) incubating the microorganismwith the above-identified compound, where nitroreductase is theactivator, for a time and under conditions sufficient for the compoundto enter the cell and be exposed to a nitroreductase if present in themicroorganism; and (b) determining whether SIG generates a greatersignal than the signal generated by the compound when not exposed to anitroreductase. In this method, a greater signal indicates that themicroorganism comprises a nitroreductase.

In additional embodiments, a method of identifying nitroreductase in asample is provided. The method comprises (a) incubating the sample withthe with the above-identified compound, where nitroreductase is theactivator, then (b) determining whether SIG generates a greater signalthan the signal generated by the compound when not exposed to anitroreductase. Here, a greater signal indicates that the samplecomprises a nitroreductase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of the mechanism of action of self-immolativeprobes of the present invention.

FIG. 2 is a graph illustrating fluorescence enhancement of compound CD-6as a function of time. The lines represent fluorescence intensity at 0,1, 5, 15 and 60 minutes after induction by sodium hydrosulfite solution.

FIGS. 3A-D are fluorescence micrographs showing compound CD-1 detectionof hypoxic HeLa cells post chemical hypoxia induction and post-anoxiatreatment, while control compound, CD-3, lacking ap-nitro group, isinactive in the live cells at the above conditions. FIG. 3A shows HeLacells that were seeded on the glass slides and treated the next day with5 μM of CD-1 (probe). Hypoxia was induced chemically as described inExample 6. After 3.5 h, cells were washed with PBS, coverslipped andobserved using a fluorescence microscope with FITC filters, 490ex/525em.FIG. 3B shows HeLa cells that were seeded on the glass slides andtreated the next day with 5 μM of CD-3 (control). Hypoxia was inducedchemically as described in Example 6. After 3.5 h, cells were washedwith PBS, coverslipped and observed using a fluorescence microscope withFITC filters, 490ex/525em. FIG. 3C shows HeLa cells that were seeded onthe glass slides and treated the next day with 5 μM of CD-1 (Probe) orCD-3 (Control). Cells were subjected to anoxic conditions (95% of N₂, 5%of CO₂) as described in Example 6. After 3.5 h, cells were washed withPBS, coverslipped and observed using fluorescence microscope with FITCfilters, 490ex/525em. FIG. 3D shows HeLa cells that were seeded on theglass slides and treated the next day with 0.2 M pimonidazole. Cellswere subjected to anoxic conditions (95% of N₂, 5% of CO₂) as describedin Example 6. After 3.5 h, cells were washed with PBS, coverslipped andobserved using a fluorescence microscope with FITC filters, 490ex/525em.

FIGS. 4A-4D are fluorescence micrographs of hypoxic HeLa cells withhypoxic probes CD-1, CD-4, CD-5 or CD-6. HeLa cells were seeded on theglass slides and treated the next day with a self-immolative hypoxiaprobe. The following probes were employed: CD-5 (5 μM, FIG. 4A), CD-1 (5μM, FIG. 4B), CD-4 (1 μM, FIG. 4C), CD-6 (1 μM, FIG. 4D). Hypoxia wasinduced chemically as described in Example 7. After 3.5 h incubation,cells were washed with PBS, coverslipped and visualized using an OlympusBX-51 fluorescence microscope with a DAPI filter set (350 ex/470em) forCD-5, an FITC filter set (490ex/525em) for CD-1, an orange filter set(550ex/620em) for CD-4 and a Texas Red filter set (596ex/670em) forCD-6.

FIG. 5 depicts histograms showing hypoxia detection in Jurkat cellsusing flow cytometry and CD-1 (panel A) and CD-6 (panel B) hypoxiaprobes.

FIGS. 6A-6C are micrographs showing multiplex detection of both cellularoxygen content and cellular redox status using hypoxia self-immolativeprobes and common ROS detecting reagents.

FIGS. 7A-7C and FIGS. 8A-8D are dot plot graphs depicting the results ofmultiplex flow cytometry assays for combined detection of hypoxia andredox status in live cells. The numbers in the quadrants of the dotplots indicate percentage of the cells.

FIG. 9 is a bar graph showing ratios of fluorescence generated inhypoxia-induced HeLa cells to fluorescence of untreated cells.

FIG. 10 is a bar graph showing luminance of compound CD-8 after varioustreatments.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Additionally, the use of “or” is intended to include“and/or”, unless the context clearly indicates otherwise.

As used herein, a “self-immolative probe” refers to a signaling moleculecovalently bound to a moiety (a “self-immolative arm”) such that theself-immolative arm inhibits the signaling molecule from signaling. Theself immolative arm is covalently bound to an enzyme substrate such thatthe action of the enzyme causes a destabilization of the self-immolativearm such that the self immolative arm becomes removed from the signalingmolecule, allowing the signaling molecule to signal.

As used herein, “hypoxic” cells are cells with inadequate oxygen. Suchcells are also “anoxic,” which refers to a complete deprivation ofoxygen.

The present invention provides probes that may be used in the detectionof specific enzyme activities or the presence of an analyte in vivo orin vitro. Structurally the probes represent signalogenic moleculescomprising a signaling molecule, SIG, functionalized with one or moreself-immolative groups, SI, and a modulator, MOD. The mechanism ofaction of these probes is illustrated in FIG. 1. The self-immolativegroup(s), SI, attached to the signal, SIG, produce a signalogenic,usually colorless, non-fluorescent or non-luminescent, compound. Thesignal SIG, defined as the colored, fluorescent or luminescent dye, isreleased from the probe by a specific chemical activator (“Trigger” inFIG. 1) that acts on MOD and causes cleavage of SI from SIG. Theactivator is, for example, an enzyme or an analyte that the probe servesto detect. The released signal molecule SIG can be detected by any meansappropriate for the specific SIG, such as UV-Vis spectroscopy,fluorescence microscopy, flow cytometry, fluorescence spectroscopy orany other method known in the art. The intensity of the signal generatedmay be quantified.

The signalogenic compounds of the present invention can be representedby the following general formula:(SIG)-(SI-MOD)_(m)wherein SIG is a signaling molecule, SI is a self-immolative structurebound to SIG such that SIG has a reduced signal relative to the signalof SIG without SI, MOD is a moiety bound to SI that is subject toenzymatic modification, and m is an integer from 1 to about 10. When MODis modified by an activator, SI is destabilized and self-cleaved fromSIG such that SIG generates an increased signal. SIG in the abovestructure can be any signaling molecule including, but not limited to, afluorophore, a chromophore, a luminescent compound etc.

In some embodiments, the compound comprises

wherein

L-Z is MOD and is ortho or para to the benzyl carbamate group, wherein

-   -   Z is a reducible nitrogen-containing group, or an amino group        with an electron-deficient moiety, and    -   L is nothing when Z is a reducible nitrogen-containing group,        otherwise L is an unsubstituted straight-chain, branched or        cyclic alkyl, alkenyl or alkynyl group, a substituted        straight-chain, branched or cyclic alkyl, alkenyl or alkynyl        group wherein one or more C, CH or CH₂ groups are substituted        with an O atom, N atom, S atom, or NH group, an unsubstituted or        substituted aromatic group, or a linear or branched sequence of        amino acids;

each R¹ is independently a hydrogen, a halogen, a Z, a cyano group (CN),an isocyano group (NC), a thiocyano group (SCN), an isothiocyano group(SNC), an azido group (N₃), a trihalomethyl group (CX₃, where X is ahalogen); a sulfonate group (SO₃R³), a sulfate group (OSO₃R³), acarboxyl group (CO₂H), a carbonyl group (COR³), an ester group (CO₂R³ orOCOR³), an amido group (CONR³ ₂ or NR³COR³), a carbamate group(NR³CO₂R³), a phosphate group (OPO₃R³ ₃), a phosphonate group (PO₃R³ ₂),an amino group (NR³ ₂), an alkoxy group (OR³), a thiol group (SR³), asulfoxy group (SOR³), a sulfone group (SO₂R³), a sulfonamide group(SO₂NR³ ₂), a phosphino group (PR³ ₂), or a silane group (SiR³ ₃);

each R³ is independently a hydrogen, an unsubstituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group, a substitutedstraight-chain, branched or cyclic alkyl, alkenyl or alkynyl groupwherein one or more C, CH or CH₂ groups are substituted with an O atom,N atom, S atom, or NH group, or an unsubstituted or substituted aromaticgroup;

n is 0, 1, 2, 3 or 4; and

R² is a hydrogen, an unsubstituted straight-chain, branched or cyclicalkyl, alkenyl or alkynyl group, a substituted straight-chain, branchedor cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH orCH₂ groups are substituted with an O atom, N atom, S atom, NH group, COgroup, OCO group or CONR³ group, or an unsubstituted or substitutedaromatic group.

In some of these embodiments, Z is a reducible nitrogen-containing groupselected from the group consisting of a nitro group (NO₂), an azo group(N═N), a hydrazo group (NH—NH), a nitroso group (NO), and ahydroxylamino group (NHOH). These embodiments are particularly usefulwhere the activator is a nitroreductase. See Examples.

In other embodiments, Z is an amino group with an electron-deficientmoiety, for example a carbonyl (C═O), a phosphoryl (PO₃ ²⁻) or asulfonyl (SO₃ ⁻) moiety.

In additional embodiments, Z is an amino group with anelectron-deficient moiety and L is an amino acid sequence that is asubstrate for an activator that is an enzyme. In these embodiments, theenzyme is capable of cleaving L from Z, leading to self-cleavage of SIand releasing SIG-NR².

The mechanism of action of these self-immolative probes can beillustrated for the specific embodiments where Z is NO₂, the activatoris a nitroreductase, and SIG is a fluorophore, as follows. When exposedto nitroreductase, the nitrobenzyl carbamate group undergoes thefollowing reaction:

In this scheme, the fluorophore probe comprising the nitrobenzylcarbamate moiety will not fluoresce due to the electron withdrawingcharacter of that moiety. The nitroreductase reduces the nitro group toan amino group, which contributes an electron to the benzyl group,causing a domino-like electron transfer through the structure, resultingin destabilization and cleavage of the nitrobenzyl carbamate moiety atthe carbamate amino group. The electron-withdrawing nature of thenitrobenzyl carbamate moiety, which resulted in a lack of fluorescenceof the fluorophore, is thus removed, allowing the fluorophore tofluoresce.

Another example of the self-immolative probes of the present inventioncomprises

wherein

L-Z is MOD and is ortho or para to the benzyl group, wherein

-   -   Z is a reducible nitrogen-containing group, or an amino group        with an electron-deficient moiety, and    -   L is nothing when Z is a reducible nitrogen-containing group,        otherwise L is an unsubstituted straight-chain, branched or        cyclic alkyl, alkenyl or alkynyl group, a substituted        straight-chain, branched or cyclic alkyl, alkenyl or alkynyl        group wherein one or more C, CH or CH₂ groups are substituted        with an O atom, N atom, S atom, or NH group, an unsubstituted or        substituted aromatic group, or a linear or branched sequence of        amino acids;

each R¹ is independently a hydrogen, a halogen, a Z, a cyano group (CN),an isocyano group (NC), a thiocyano group (SCN), an isothiocyano group(SNC), an azido group (N₃), a trihalomethyl group (CX₃, where X is ahalogen); a sulfonate group (SO₃R³), a sulfate group (OSO₃R³), acarboxyl group (CO₂H), a carbonyl group (COR³), an ester group (CO₂R³ orOCOR³), an amido group (CONR³ ₂ or NR³COR³), a carbamate group(NR³CO₂R³), a phosphate group (OPO₃R³ ₃), a phosphonate group (PO₃R³ ₂),an amino group (NR³ ₂), an alkoxy group (OR³), a thiol group (SR³), asulfoxy group (SOR³), a sulfone group (SO₂R³), a sulfonamide group(SO₂NR³ ₂), a phosphino group (PR³ ₂), or a silane group (SiR³ ₃);

each R³ is independently a hydrogen, an unsubstituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group, a substitutedstraight-chain, branched or cyclic alkyl, alkenyl or alkynyl groupwherein one or more C, CH or CH₂ groups are substituted with an O atom,N atom, S atom, or NH group, or an unsubstituted or substituted aromaticgroup; and

n is 0, 1, 2, 3 or 4.

As with the previously described probes, in some embodiments of theseprobes, Z is a reducible nitrogen-containing group selected from thegroup consisting of a nitro group (NO₂), an azo group (N═N), a hydrazogroup (NH—NH), a nitroso group (NO), and a hydroxylamino group (NHOH).Such probes are useful for detecting a nitroreductase, which would serveas an activator of the probe. In other embodiments, Z is an amino groupwith an electron-deficient moiety selected from the group consisting ofcarbonyl (C═O), phosphoryl (PO₃ ²⁻) and sulfonyl (SO₃ ⁻). In additionalembodiments, Z is an amino group with an electron-deficient moiety and Lis an amino acid sequence that is a substrate for an activator that isan enzyme, wherein the enzyme is capable of cleaving L from Z, leadingto self-cleavage of SI and releasing SIG-NR².

Still another example of a self-immolative probe of the presentinvention is a compound comprising the structure

wherein

S—S is MOD;

each R² is independently a hydrogen, an unsubstituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group, a substitutedstraight-chain, branched or cyclic alkyl, alkenyl or alkynyl groupwherein one or more C, CH or CH₂ groups are substituted with an O atom,N atom, S atom, NH group, CO group, OCO group or CONR³ group, or anunsubstituted or substituted aromatic group; and

q is an integer from 1 to 4.

These probes are particularly useful for detecting disulfide reducingagents, which reduce the disulfide bond, inducing cyclization with arelease of a thiolactone and resulting in destabilization and cleavageof the self-immolative moiety to leave the active signaling moleculeSIG-NR². Nonlimiting examples of disulfide reducing agents areglutathione, cysteine, and homocysteine. Thus, this probe can detect anyof those compounds.

In some embodiments of the generalized probe(SIG)-(SI-MOD)_(m)m>1; in other embodiments, m=1. In some applications, m is preferably 1,so that only one self-immolative group needs to be removed to achievefull signal intensity of the signalophore, SIG. When one self-immolativegroup is desired but the fluorophore has more than one moiety where theself-immolative group can be attached, a blocker group that preferablydoes not substantially interfere with the signal, SIG, can be bonded toany reactive moiety on SIG where the self-immolative group is notdesired.

Thus, in some embodiments, SIG further comprises at least one blockermoiety that blocks sites of potential SI-MOD attachment during synthesisof the compound, wherein the moiety does not substantially interferewith the SIG signal.

An example of a useful blocker moiety is the urea moiety R⁴ ₂N—CO—NR⁴,where the compound is

wherein each R⁴ is independently a hydrogen, an unsubstitutedstraight-chain, branched or cyclic alkyl, alkenyl or alkynyl group, asubstituted straight-chain, branched or cyclic alkyl, alkenyl or alkynylgroup wherein one or more C, CH or CH₂ in any of the foregoing groupscan be substituted with an O atom, N atom, S atom, NH group, CO group,OCO group or CONR³ group, an unsubstituted aromatic group or asubstituted aromatic group. In some of these embodiments, two or more R⁴groups are fused to form a ring, the ring comprising one or moreheteroatoms, wherein the heteroatoms are the same heteroatoms ordifferent heteroatoms. An example of such a blocker moiety is

See, e.g., Example 1.

The signalogenic probes of the formula (SIG)-(SI-MOD)_(m) in thisinvention can be prepared by any means known in the art, for example byreacting the signal molecule, SIG, with self-immolative group(s), SI, aswell as optionally with blocker groups. In some embodiments, the signalmolecule reacts with one or more optional blocker groups and thenundergoes the reaction with one or more self-immolative groups to give amolecule (SIG)-(SI-MOD)_(m). The molecule (SIG)-(SI-MOD)_(m) has asubstantially lower signal intensity (fluorescence, luminescence orcolor intensity) than SIG optionally substituted with the blocker.However, the reactions leading to attachment of self-immolative groupsas well as blocker groups to SIG can be performed in any order. Thisorder is conveniently determined by the reaction type and nature of thesignal molecule, and can be determined by the skilled artisan withoutundue experimentation.

The signal, SIG, can be any chemical compound that has decreasedfluorescence, luminescence or color intensity when functionalized withone or more self-immolative groups, SI, and increased fluorescence,luminescence or color intensity when at least one of theseself-immolative group is removed. Preferably, SIG should benon-fluorescent, non-luminescent and colorless when SI is attached andintensely fluorescent, luminescent or colored when SI is removed.Additionally, SIG should contain or should be readily modified tocontain reactive functionalities, as further discussed below, to whichboth self-immolative and optional blocker moieties could be attached toform a probe.

The invention is not narrowly limited to the use of any particular SIG.In various embodiments, SIG is a chromophore, a fluorophore, aluminescent moiety, an enzyme, a catalytic antibody, a ribozyme or apro-enzyme.

In some embodiments, SIG is a fluorophore. Any fluorophore now known orlater discovered can be utilized in these compounds. Examples of usefulfluorophores include without limitation a symmetric or asymmetriccyanine dye, a merocyanine dye, a styryl dye, an oxazine dye, a xanthenedye, a coumarin dye or an iminocoumarin dye.

One class of the signal molecule, SIG, useful in the invention has axanthene backbone shown in Scheme I below. The structures include bothclassical xanthene dyes and their lactone forms (Structures A and B,respectively) as well as aphenylic counterparts, which have theirappended phenyl ring missing (Structures C).

The substituent R⁵ in Scheme I represents a variety of functionalitieswhere at least one R⁵ is a reactive group, which allows the attachmentof the self-immolative group SI and, if desired, at least one other R⁵is a reactive group, which allows the attachment of a blocker moiety.The R⁵s may be structurally the same or different and there may beseveral of them per ring. Also, some of the rings may not have any R⁵sattached. Suitable examples of R⁵ include, but are not limited tohydrogen, a halogen (F, Cl, Br, I), a nitro group (NO₂), a nitroso group(NO), a hydroxylamino group (NHOH), a cyano group (CN), an isocyanogroup (NC), a thiocyano group (SCN), an isothiocyano group (SNC), anazido group (N₃), a trihalomethyl group (CX₃, where X is a halogen), asulfonate group (SO₃R⁶), a sulfate group (OSO₃R⁶), a carboxyl group(CO₂H), a carbonyl group (COR⁶), an ester group (CO₂R⁶ or OCOR⁶), anamide group (CONR⁶ ₂ or NR⁶COR⁶), a carbamate group (NR⁶CO₂R⁶ or OCONR⁶₂), a phosphate group (OPO₃R⁶ ₃), a phosphonate group (PO₃R⁶ ₂), anamino group (NR⁶ ₂), an alkoxy group (OR⁶), a thiol group (SR⁶), asulfoxy group (SOR⁶), a sulfone group (SO₂R⁶), a sulfonamide group(SO₂NR⁶ ₂), a phosphino group (PR⁶ ₂), a silane group (SiR⁶ ₃), anoptionally substituted straight-chain, branched or cyclic alkyl, alkenylor alkynyl group wherein one or more C, CH or CH₂ groups can be replacedwith O atom, N atom, S atom, NH group, CO group, OCO group, CONR⁶ group,or an optionally substituted aromatic group. In these embodiments, eachR⁶ is independently hydrogen, an optionally substituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group wherein one or moreC, CH or CH₂ groups can be replaced with O atom, N atom, S atom, NHgroup, CO group, OCO group, CONR⁶ group, or an optionally substitutedaromatic group.

Two or more R⁵ groups in these fluorophores can be linked together toform rings containing one or more of the same or different heteroatoms,such as O, N or S.

Substituents R⁵ in these fluorophores that are not directly involved inattachment of self-immolative or urea-containing groups may be presentin the molecule for other reasons. These reasons may includemodification of spectroscopic characteristics of the dye, itssolubility, chemical stability or photobleaching resistance. Somesubstituents R⁵ may be useful for binding to a biomolecule or structureto be studied, such as nucleic acid, protein or lipid.

As discussed above, one of the R⁵ or R⁶ groups is, or can be substitutedto contain, a reactive group thereby allowing the dyes of the presentinvention to be attached to an SI-MOD group. Examples of reactive groupsthat may find use in the present invention can include but not belimited to a nucleophilic reactive group, an electrophilic reactivegroup, a terminal alkene, a terminal alkyne, a platinum coordinate groupor an alkylating agent.

There are a number of different electrophilic reactive groups that mayfind use in these embodiments. Examples include but not be limited toisocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine,4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine,mono- or di-halogen substituted diazine, maleimide, haloacetamide,aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol,azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal and aldehyde groups.Nucleophilic reactive groups can include but not be limited to reactivethiol, amine and hydroxyl groups. For purposes of synthesis of dyes,reactive thiol, amine or hydroxyl groups can be protected during varioussynthetic steps and the reactive groups generated after removal of theprotective group.

One class of xanthene fluorophores useful in the present inventionincludes but not limited to rhodamine and rhodamine derivatives, such asPennsylvania Green, Tokyo Green, Oregon Green, Singapore Green, androsamines and rhodols and their derivatives. Some of these derivativesare shown below in Scheme II. The rhodamine, rosamine and rhodolbackbone structures can be extended by adding additional rings as shownin Scheme III, or their appended phenyl ring might be missing to formaphenylic counterparts.

Another class of fluorescent dyes pertinent to the present invention isderivatized from the aforementioned rhodamines, rosamines and rhodolsand can be represented by the general structures shown in Scheme IV.

The substituent R⁵ in Scheme IV is defined as described for Scheme I.The moiety A can be oxygen or sulfur while Z can be oxygen, sulfur ornitrogen unsubstituted or substituted with a group Y. The group Y, inturn, can be hydrogen, an optionally substituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group wherein one or moreC, CH or CH₂ groups can be replaced with O atom, N atom, S atom, NHgroup, CO group, OCO group, CONR³ group, or an optionally substitutedaromatic group. Y can also be another nitrogen, oxygen or sulfur atomsubstituted with hydrogen or an optionally substituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group wherein one or moreC, CH or CH₂ groups can be replaced with O atom, N atom, S atom, NHgroup, CO group, OCO group, CONR³ group, or an optionally substitutedaromatic group. The substituent, Y, can be a part of an aliphatic oraromatic cyclic structure such as morpholine, piperidine, pyrrolidine,piperazine, imidazole, triazole, oxazole, thiazole and others known inthe art. Additionally, both Z and Y can contain electrophilic ornucleophilic reactive groups defined above.

Yet another class of fluorescent dyes pertinent to the present inventionis based on coumarin and iminocoumarin backbone structure shown inScheme V.

The substituent R⁵ in the Scheme V represents functionalities defined inScheme I above while A can be oxygen atom, O, or imino group, NH. Someof the compounds in this category are shown below in Scheme VI. Thebackbone structure can be extended by adding additional rings, aliphaticor aromatic, substituted or unsubstituted.

In other embodiments of the compounds of the present invention, SIG is aluminescent moiety. Any luminescent moiety, including anychemiluminescent or bioluminescent moieties, now known or laterdiscovered, can be utilized in these embodiments. In some aspects ofthese embodiments, the compound comprises the structure:

wherein

each R⁵ is independently hydrogen, a halogen (F, Cl, Br, I), a nitrogroup (NO₂), a nitroso group (NO), a hydroxylamino group (NHOH), a cyanogroup (CN), an isocyano group (NC), a thiocyano group (SCN), anisothiocyano group (SNC), an azido group (N₃), a trihalomethyl group(CX₃, where X is a halogen); a sulfonate group (SO₃R⁶), a sulfate group(OSO₃R⁶), a carboxyl group (CO₂H), a carbonyl group (COR⁶), an estergroup (CO₂R⁶ or OCOR⁶), an amide group (CONR⁶ ₂ or NR⁶COR⁶), a carbamategroup (NR⁶CO₂R⁶ or OCONR⁶ ₂), a phosphate group (OPO₃R⁶ ₃), aphosphonate group (PO₃R⁶ ₂), an amino group (NR⁶ ₂), an alkoxy group(OR⁶), a thiol group (SR⁶), a sulfoxy group (SOR⁶), a sulfone group(SO₂R⁶), a sulfonamide group (SO₂NR⁶ ₂), a phosphino group (PR⁶ ₂), asilane group (SiR⁶ ₃), an optionally substituted straight-chain,branched or cyclic alkyl, alkenyl or alkynyl group wherein one or moreC, CH or CH₂ groups can be replaced with O atom, N atom, S atom, NHgroup, CO group, OCO group, CONR⁶ group, or an optionally substitutedaromatic group; and

each R⁶ is independently hydrogen, an optionally substitutedstraight-chain, branched or cyclic alkyl, alkenyl or alkynyl groupwherein one or more C, CH or CH₂ groups can be replaced with O atom, Natom, S atom, NH group, CO group, OCO group, CONR⁶ group, or anoptionally substituted aromatic group.

The activator for the compounds of the present invention can be anychemical that can modify MOD such that SI is destabilized andself-cleaved from SIG. For example, as discussed above, the activatorfor some SI-MOD embodiments can be a disulfide reducing agent such asglutathione, cysteine, or homocysteine. In other embodiments, theactivator is an enzyme.

Where the activator is an enzyme, the invention is not limited to anyparticular enzyme, as it is believed that a MOD can be designed for anyenzyme without undue experimentation. Nonlimiting examples ofenzyme-activators that can be utilized for these embodiments includenitroreductases, kinases, aminopeptidases, esterases, lipases,proteases, peptidases, phosphatases, sulfatases, sulfotransferases,carboxylases, decarboxylases, glycosylases, amidases, deamidases,aminases, deaminases, acetyltransferases, methylases, deacetylases,demethylases, and acetylases.

In some embodiments, the enzyme activator is an esterase or a lipase.Exemplary compounds useful for esterase or lipase activation comprisethe structure:

wherein

q is an integer from 1 to 4,

X is an oxygen or sulfur, and

R⁷ is an unsubstituted straight-chain, branched or cyclic alkyl, alkenylor alkynyl group, a substituted straight-chain, branched or cyclicalkyl, alkenyl or alkynyl group wherein one or more C, CH or CH₂ in anyof the foregoing groups can be substituted with an O atom, N atom, Satom, NH group, CO group, OCO group or CONR³ group, an unsubstitutedaromatic group or a substituted aromatic group.

In more particular embodiments, the compound comprises the structure:

These compounds are useful for esterase or lipase detection, where theenzyme cleaves the thioester bond, causing spontaneous self-immolativecollapse of the SI moiety and concomitant release of SIG.

In other embodiments, the enzyme is a nitroreductase. Exemplarycompounds that are useful for nitroreductase activation comprise thestructures

Specific examples of such compounds include

In these embodiments, when the probe is activated by nitroreductase orthe enzymes that have nitroreductase activity, the nitro group(s) getsreduced to amino- and hydroxylamino functionalities. This conversiontriggers a self-immolative decomposition of the probe with a release ofSIG.

Nitroreductases include a broad family of enzymes that reducenitrogen-containing compounds including those containing the nitrofunctional group. Members of this family utilize FMN as a cofactor andare often found to be homodimers. Nitroreductases include enzymes from:EC 1.1: which includes oxidoreductases that act on the CH—OH group ofdonors, EC 1.2: which includes oxidoreductases that act on the aldehydeor oxo group of donors, EC 1.3: which includes oxidoreductases that acton the CH—CH group of donors, EC 1.4: which includes oxidoreductasesthat act on the CH—NH₂ group of donors, EC 1.5: which includesoxidoreductases that act on CH—NH group of donors, E C 1.6: whichincludes oxidoreductases that act on NADH or NADPH, EC 1.7: whichincludes oxidoreductases that act on other nitrogenous compounds asdonors, EC 1.8: which includes oxidoreductases that act on a sulfurgroup of donors, EC 1.9: which includes oxidoreductases that act on aheme group of donors, EC 1.10: which includes oxidoreductases that acton diphenols and related substances as donors, EC 1.11: which includesoxidoreductases that act on peroxide as an acceptor (peroxidases), EC1.12: which includes oxidoreductases that act on hydrogen as donors, EC1.13: which includes oxidoreductases that act on single donors withincorporation of molecular oxygen (oxygenases), EC 1.14: which includesoxidoreductases that act on paired donors with incorporation ofmolecular oxygen, EC 1.15: which includes oxidoreductases that act onsuperoxide radicals as acceptors, EC 1.16: which includesoxidoreductases that oxidize metal ions, EC 1.17: which includesoxidoreductases that act on CH or CH2 groups, EC 1.18: which includesoxidoreductases that act on iron-sulfur proteins as donors, EC 1.19:which includes oxidoreductases that act on reduced flavodoxin as adonor, EC 1.21: which includes oxidoreductases that act on X—H and Y—Hto form an X—Y bond, and EC 1.97: which includes other oxidoreductases.Specific examples of nitroreductases include DT-diaphorase [NQO1;E.C.1.6.99.2]; cytochrome P450-reductase [CYPOR; E.C.1.6.2.4]; induciblenitric oxide synthase [NOS2A; E.C.1.14.13.39]; cytochrome B5 reductase[DIAL; E.C.1.6.2.2]; xanthine oxidase [XO; E.C.1.17.3.2]; xanthinedehydrogenase [XDH; E.C.1.17.1.4]; adrenodoxin oxidoreductase [FDXR;E.C.1.18.1.2]; methionine synthase reductase [MTRR; E.C.1.16.1.8];aldose reductase [ALDR1; E.C.1.1.1.21]; aldehyde reductase [AKR1B10;E.C.1.1.1.2] and thioredoxin reductase [TXNRD; E.C.1.8.1.9]. Thus, thecompounds of these embodiments can be utilized to detect any of theabove enzymes.

In additional embodiments, the enzyme is a protease or peptidase and thecompound comprises the structure:SIG-(SI-AA₁-AA₂ . . . AA_(n−1)-AA_(n))_(m)

wherein each AA is independently an amino acid, n is an integerrepresenting the total number of amino acids, wherein n is from 1 toabout 200, and m is an integer from 1 to about 10; and

wherein the protease or peptidase is capable of removing the amino acidsequence, allowing self-cleavage of the SI. Thus, although more than oneamino acid is depicted in the above structure, these embodimentsencompass the structures where there is only 1 amino acid (n=1). It isnoted that in these embodiments, the amino acid sequence must be onethat is a substrate for the particular protease or peptidase to bedetected. Thus, the amino acid sequence for these compounds variesaccording to the specific requirements of the assayed protease orpeptidase.

In more specific embodiments, the compound for detecting a protease orpeptidase comprises the structure:

In a modification of the above peptidase-detecting embodiments,compounds are provided for detecting enzymes or non-enzymatic processesinvolved in addition or removal of a posttranslational modification of aprotein. Nonlimiting examples of such enzymes include enzymes involvedin acylation, alkylation, amidation, amino acid addition, diphthamideformation, gamma-carboxylation, glycosylation, glypiation, addition of aheme moiety, hydroxylation, iodination, attachment of nucleotide moiety,nitrosylation, S-glutathionylation, oxidation, phosphopantetheinylation,phosphorylation, pyroglutamate formation, sulfation, selenoylation,SUMOylation, or ubiquitination.

In some embodiments, the compound for detecting enzymes involved in aposttranslational modification comprises the structure:

wherein each AA is independently an amino acid, PTM is apost-translational modification on any of the amino acids, n is aninteger representing the total number of amino acids, wherein n is from1 to about 200, and m is an integer from 1 to about 10; and

wherein the enzyme involved in post-translational modification iscapable of adding or removing the PTM, and, when the PTM is removed, aprotease or peptidase is capable of removing the amino acid sequence,allowing self-cleavage of the SI. Thus, the detection of the enzymeinvolved in post-translational modification is a two-step process. Inthe first step, the enzyme, if present, adds or removes the PTM; in thesecond step a protease or peptidase is added, where, if thepost-translational modification is not present, the protease orpeptidase is able to remove the amino acid(s), releasing SIG, whereas ifthe post-translational modification is present, the protease orpeptidase is unable to remove the amino acid(s) and SI remains bound toSIG, preventing the signal from being observed.

In more specific embodiments, the compound comprises the structure:

In a specific post-translational modification, the enzyme is a proteinkinase. One structure useful for these embodiments comprises

wherein —OH is a hydroxyl moiety on a serine, threonine, or tyrosinethat is a target for phosphorylation by the kinase. It is noted that theamino acid sequence utilized in these embodiments must be one that isrecognized by the particular protein kinase that is being detected.

In these embodiments, upon exposure to a protein kinase and ATP, thehydroxyl moiety becomes phosphorylated. With phosphorylation of thecompound and subsequent addition of a protease or peptidase, the aminoacids cannot be cleaved by the protease or peptidase, and SIG remainsbound to SI, resulting in no signaling, whereas without phosphorylationof the compound, the protease or peptidase is able to remove the aminoacids, resulting in the self-immolative removal of SI and release of aSIG that provides a signal. Thus, the assay associated with thiskinase-detecting structure is a negative assay, since lack of kinaseaction results in a signal, whereas the presence of the kinase resultsin no signal.

More specific embodiments of the kinase-detecting compound comprise thestructures:

In another specific post-translational modification, the enzyme is aphosphatase. An exemplary compound for detecting the phosphatasecomprises the structure:

wherein —OPO₃ ²⁻ is a phosphate moiety on a serine, threonine, ortyrosine that is a target for dephosphorylation by the phosphatase. Itis noted that the amino acid sequence utilized in these embodiments mustbe one that is recognized by the particular phosphatase that is beingdetected. Such a phosphatase is detected with these compounds bycombining the compound with a sample to be tested for the phosphatasethen adding a protease or peptidase that is capable of removing theamino acids in the absence, but not in the presence, of the phosphatemoiety. Thus, if the phosphatase is present in the sample, the phosphategroup will be removed, allowing the protease or peptidase to remove theamino acids, resulting in the self-immolative removal of SI and releaseof a SIG that provides a signal. However, without a phosphatase in thesample, the phosphate group remains, preventing the protease orpeptidase from removing the amino acids. SIG then remains bound to SI,resulting in no signaling.

More specific embodiments of the phosphatase-detecting compound comprisethe structures:

In yet another specific post-translational modification, the enzyme is ahistone deacetylase (HDAC). An exemplary compound for detecting the HDACcomprises the structure:

An HDAC is detected with these compounds by combining the compound witha sample to be tested for the HDAC then adding a protease or peptidasethat is capable of removing the remaining amino acid in the absence, butnot in the presence, of the acetyl moiety on the ε-N-acetyl moiety.Thus, if the HDAC is present in the sample, the acetyl group will beremoved, allowing the protease or peptidase to remove the amino acids,resulting in the self-immolative removal of SI and release of a SIG thatprovides a signal. However, without an HDAC in the sample, the acetylgroup remains, preventing the protease or peptidase from removing theamino acids. SIG then remains bound to SI, resulting in no signaling.

More specific embodiments of the HDAC-detecting compound comprise thestructures:

Examples of particular compounds that detect HDAC are

The above compounds are useful in methods of detecting an activator.Thus, in some embodiments, a method of determining whether a samplecomprises an activator is provided. The method comprises

(a) incubating the sample with the above-identified compound for a timeand under conditions sufficient for MOD to be modified by the activator;and

(b) determining whether SIG generates a greater signal than the signalgenerated by the compound without the activator. In these embodiments, agreater signal indicates that the sample comprises the activator.

These methods are useful for detection of the activator in any sample.In some embodiments, the sample is a fluid of an organism or a colony oforganisms, or an extract thereof. In some aspects of these embodiments,the organism or colony of organisms is microorganisms, for example aprokaryote or an archaea, or a eukaryotic microorganism such as aprotist. In other aspects, the organism is a multicellular eukaryote. Insome of these embodiments, the sample is an extract of a cell, tissue ororgan of the multicellular organism. The eukaryote multicellularorganism can be a mammal or any other eukaryote.

In some embodiments, the sample for these methods comprises a livingcell, i.e., a prokaryotic, archaeal or eukaryotic cell, e.g., from amammal, for example a human.

The activator for these methods can be any activator capable of actingon MOD and initiating the self-immolative cleavage of the SI from SIG.In some embodiments, the activator is a disulfide reducing agent.Exemplary compounds for detecting such an activator is provided above.Particular disulfide reducing agents that can act as activators includeglutathione, cysteine or homocysteine.

In various embodiments of these methods, the activator is an enzyme,e.g., a nitroreductase, a kinase, an aminopeptidase, an esterase, alipase, a protease, a peptidase, a phosphatase, a sulfatase, asulfotransferase, a carboxylase, a decarboxylase, a glycosylase, anamidase, a deamidase, an aminase, a deaminase, an acetyltransferase, amethylase, a deacetylase, a demethylase, or an acetylase.

In some of these methods, the enzyme is a lipase or esterase. Exemplarycompounds useful to detect a lipase or esterase are provided above.

In other embodiments of these methods, the enzyme is a nitroreductase.Exemplary compounds for detecting nitroreductases are also providedabove.

In additional embodiments of these methods, the enzyme is a protease orpeptidase. Examples of compounds useful for detecting proteases arefurther provided above.

The enzyme for these methods can also be an enzyme involved in additionor removal of a posttranslational modification of a protein. See abovefor exemplary compounds useful for these methods. In one aspect of theseembodiments, the enzyme is a protein kinase. As discussed above, thesample is also incubated with a peptidase either during or after theincubation step (a), but before the determining step (b). As furtherdiscussed above, the signal in these methods is an inverse signal suchthat increased activation of SIG indicates a decrease in the amount ofprotein kinase in the sample. In another aspect of these embodiments,the enzyme is a phosphatase. As discussed above, the sample is alsoincubated with a peptidase either during or after the incubation step(a), but before the determining step (b). In an additional aspect ofthese embodiments, the enzyme is a histone deacetylase. As alsodiscussed above in relation to the histone deacetylase compounds, thesample is also incubated with a peptidase either during or after theincubation step (a), but before the determining step (b).

Also provided herewith is a method of determining whether a cellcomprises a nitroreductase. The method comprises

(a) incubating the cell with any of the above-described compounds thatare useful for detecting a nitroreductase, for a time and underconditions sufficient for the compound to enter the cell and be exposedto a nitroreductase if present in the cell; and

(b) determining whether SIG generates a greater signal than the signalgenerated by the compound when not exposed to a nitroreductase. In thesemethods, a greater signal indicates that the cell comprises thenitroreductase. Examples of these assays are described in Examples 6-12below.

In these methods, the cell can be incubated with the compound for anylength of time, e.g., more than about 120 minutes, about 120 minutes orless, about 60 minutes or less, or about 30 minutes or less. Shorterincubation times (e.g., 10 minutes or less, 5 minutes or less, or 2minutes or less) are sufficient where the nitroreductase is not in acell. See, e.g., FIG. 2.

The cell in these embodiments can be any cell of any microorganism, forexample a mammalian cell, or the cell of a microorganism, for example abacterium.

This method can be used to identify any enzyme having nitroreductaseactivity, including DT-diaphorase [NQO1; E.C.1.6.99.2]; cytochromeP450-reductase [CYPOR; E.C.1.6.2.4]; inducible nitric oxide synthase[NOS2A; E.C.1.14.13.39]; cytochrome B5 reductase [DIAL; E.C.1.6.2.2];xanthine oxidase [XO; E.C.1.17.3.2]; xanthine dehydrogenase [XDH;E.C.1.17.1.4]; adrenodoxin oxidoreductase [FDXR; E.C.1.18.1.2];methionine synthase reductase [MTRR; E.C.1.16.1.8]; aldose reductase[ALDR1; E.C.1.1.1.21]; aldehyde reductase [AKR1B10; E.C.1.1.1.2] orthioredoxin reductase [TXNRD; E.C.1.8.1.9].

In various embodiments of these methods, SIG is a chemiluminescent dyeor a fluorophore. Particular compounds useful for these methods include

This method can also be used with microorganisms, e.g., bacteria, todetermine whether cells of the microorganism produce nitroreductase.Such methods are useful in identifying or characterizing microorganisms,e.g., that are in a tissue or fluid sample of a vertebrate infected withthe microorganism or in an environmental sample.

Additionally, this method can be used to identify nitroreductase in asample, by incubating the sample with any of the above-identifiedcompounds, then determining whether fluorescence or luminescence of thecompound increases during the incubation. In this method, an increase influorescence or luminescence during the incubation indicates that thesample comprises a nitroreductase. In some embodiments, thenitroreductase is quantified in the sample by comparing the fluorescenceor luminescence of the compound after the incubation with fluorescenceor luminescence of a known quantity of nitroreductase incubated with thecompound under the same conditions.

These methods for detecting nitroreductase in cells are particularlyuseful for detecting hypoxia in cells, for example in tumor cells. Thetumor microenvironment is one of the most critical factors in tumorprogression and cancer treatment outcome. The presence of eithertransiently or chronically hypoxic cells constitutes an importantcharacteristic of solid tumors since low oxygen levels are not usuallypresent in tissues under physiological conditions. A correlation existsbetween the percentage of hypoxic cells in the solid tumor and cancertreatment prognosis since hypoxic cells are refractory to radiationtherapy and resistant to toxic drugs used in chemotherapy. As a result,detection and analysis of hypoxic cell fractions in tumors can provideinvaluable information about cancer status, its prognosis and insightinto the specific treatment options.

Detection of hypoxic cells also plays a role in research areas outsideof cancer. Such areas include studies of reactive oxygen and nitrogenspecies, ageing, apoptosis, autophagy, cardiac and pulmonary ischemia,neurodegenerative and immunological disorders.

Various techniques have been used to measure cell oxygenation status.Some of those techniques, such as oxygen microelectrodes,histomorphometric analysis or determination of DNA strand breaks, areinvasive and/or use equipment not readily available for investigators.Other techniques are based on the hypoxia-induced reduction of a labeled2-nitroimidazole. Labels used in this context include ¹⁴C, ³H, ¹⁹F,⁷⁵Br, ⁷⁶Br and ⁷⁷Br employed in NMR, PET, autoradiography andimmunohistochemistry. Fluorescent or luminescent dyes are an attractivealternative option since they are sensitive, environmentally safe andthey can be used in non-invasive assays. As is known, mammaliannitroreductases require anaerobic conditions for activity. Thus,fluorescent or luminescent dyes that are activated by nitroreductaseprovide a good approach for measuring oxygen status of mammalian cells.

Besides measurement of hypoxia fluorescent or luminescent dyes activatedby nitroreductase can be employed in order to detect several pathogenicmicroorganisms producing nitroreductases. Additionally, the ability ofnitroreductase to reduce nitro groups has been exploited as part of areporter gene assay that employs the red carbocyanine dye CytoCy5S. Thissquaraine carbocyanine structure contains a 3,5-dinitrophenylsubstituent that essentially quenches the fluorescence at longwavelengths. Upon enzyme activity, however, the nitro-groups are reducedto hydroxylamines (and presumably amino-groups) intracellularly. Thisrelieves the quenching and results in an increase in fluorescence. Thesubstrate has been further modified to make it into the di-ethyl esterfor membrane permeability. These ethyl esters are removed byintracellular esterase activity so that the fluorescent end-product iswell retained within the cell. The assay therefore uses this‘red-shifted’ excitation and emission (excitation 647 nm emission 667nm) for the reporter gene assays which allows use with other fluorescentreporters, such as green fluorescent protein (GFP), to be used in thesame cell. Expression of nitroreductase has been demonstrated in anumber of mammalian cells without reported toxicity.

For a fluorescent marker to be suitable for determining hypoxicconditions, it should meet several conditions. First, it should havefavorable excitation and emission wavelengths, and as a result beexcitable and detectable by readily available light sources and filtersystems. Second, it should have a high quantum yield and high molarabsorption coefficient. Third, it should exhibit a significantfluorescence difference between the hypoxic and the normoxic forms ofthe dye to maximize the signal-to-background ratio.

Several dyes have been described for which the fluorescence increasesupon the activation by nitroreductases. US Patent PublicationUS2002/0031795 A1 describes a group of non-fluorescent 7-nitrocoumarinsthat are reduced by nitroreductase to fluorescent species, and are usedfor the detection of microbial infection. Additional nitrocoumarincompounds are provided in US Patent Publication US2010/0173332. Also,PCT Publication WO 2008/030120 describes chemically diversifiednon-fluorescent probes that are reduced in the presence ofnitroreductase to form fluorescent derivatives. Additionally, afluorogenic substrate which can detect the activity of certain enzymesthat reduce nitro compounds to amines and inorganic nitrates,6-Chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine (“CNOB”, Invitrogen) hasbeen developed. Although the compound is a good substrate for somebacterial nitroreductases it is apparently not a substrate for mammaliancounterparts. The compound lacks stability in culture medium underconditions of low oxygen, making it unsuitable as a probe for mammaliansingle-electron reductases which require anaerobic conditions foractivity. Nitro group quenched cyanine dyes are also taught in US PatentPublication US2003/0186348A1, U.S. Pat. Nos. 7,579,140 B2, and 7,662,973B2. Those dyes detect microbial nitroreductases in connection withreporter gene applications. The above-described compounds, however,generally have considerable fluorescence in their quenched, unreducedform. Upon the action of nitroreductase a modest 3-4-fold enhancement ofthe fluorescence is observed, offering a limited dynamic range ofquantification.

Nitro-substituted squaraine reporter dyes and the methods of using suchdyes for detection of nitroreductase enzyme activity and nitroreductasegene expression in cellular assays are disclosed in US PatentPublication US2008/0317674. However, the majority of the compoundsdescribed therein contain a nitroreductase-sensing nitrobenzyl appendagewhich is non-conjugated to the dye structure, and therefore generaterather modest fluorescence enhancement upon activation by the enzyme.

Thus, the present invention is also directed to a method of determiningwhether a mammalian cell is hypoxic. The method comprises (a) incubatingthe cell with the above-identified compound for a time and underconditions sufficient for the compound to enter the cell and be exposedto a nitroreductase if present in the cell, where the nitroreductase isindicative of hypoxia in the cell; and (b) determining whether thesignal of the compound in the cell increases during the incubation. Inthis method, an increase in the signal intensity during the incubationindicates that the cell is hypoxic.

When using this method with cells, e.g., mammalian cells to determinewhether the cells are hypoxic, the method can be combined with the useof dyes for determining other characteristics of the cell. In some ofthese embodiments, oxidative stress in the cell is also determined, byincluding a probe that detects reactive oxygen species in the incubationstep (a), then determining whether the probe detects reactive oxygenspecies in the cell, where the presence of reactive oxygen speciesindicates oxidative stress in the cell. Any probe that detects reactiveoxygen species can be used here, for example 2′,7′-dichlorofluoresceindiacetate, dihydrorhodamine 123, 3′-(p-aminophenyl) fluorescein (APF),3′-(p-hydroxyphenyl) fluorescein (HPF), aminophenoxycalcein (APC),mitoAR, mitoHR, DPAX, DMAX, dihydroethidium, or the probes described inU.S. Patent Publications US2010/0081159 and 2009/0253118. See alsoTarpey et al., 2004; and Nagano, 2009. Examples of reactive oxygenspecies that can be detected by these methods include superoxide (O₂^(•−)), hydroperoxy (HO^(•) ₂), hydrogen peroxide (H₂O₂), peroxynitrite(ONOO⁻), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxylradical (HO^(•)), peroxy radical (ROO^(•)), alkoxy radical (RO^(•)),singlet oxygen (¹O₂), lipid peroxides, lipid peroxyradicals or lipidalkoxyl radicals, and combinations thereof.

In other embodiments, the method for determining nitroreductase activityand/or hypoxia in a cell further comprises determining the nitrativestress in the cell, by including a probe that detects reactive nitrogenspecies in the incubation step (a), then determining whether the probedetects reactive nitrogen species in the cell, where the presence ofreactive nitrogen species indicates nitrative stress in the cell.Examples of useful probes for this method include diaminoanthraquinone,diaminonaphthalene, a diaminofluorescein, a diaminorhodamine, adiaminocyanine, an NiSPY, dichlorodiaminocalcein, DAMBO-P^(H) and theprobes described in U.S. Patent Publications US2010/0081159 and2009/0253118. See also Ueno et al., 2006. Examples of reactive nitrogenspecies that can be detected in these methods include nitric oxide (NO),nitrogen dioxide radical (^(•)NO₂), peroxynitrite anion (ONOO⁻),peroxynitrous acid (ONOOH), nitrosoperoxycarbonate anion (ONOOCO₂ ⁻),nitronium cation (NO₂ ⁺), nitrosonium cation (NO⁺) or dinitrogentrioxide (N₂O₃), and combinations thereof.

In additional embodiments, the method for determining nitroreductaseand/or hypoxia in a cell further comprises determining the halogenatingstress in the cell, by including a probe that detects reactive halogenspecies in the incubation step (a), then determining whether the probedetects reactive halogen species in the cell, where the presence ofreactive halogen species indicates halogenating stress in the cell.Examples of such probes include those described in U.S. PatentPublications US2010/0081159 and 2009/0253118, and Matthew and Anastasio,2006; and Anastasio and Matthew, 2006. Examples of reactive halogenspecies that can be detected by these methods include hypochlorous acid(HOCl), hypochlorite ion (ClO⁻) monochloramine (NH₂Cl), chlorine dioxide(ClO₂), nitryl chloride (NO₂Cl), chlorine (Cl₂), bromine (Br₂),bromochloride (BrCl), hypobromous acid (HOBr), hypobromite ion (BrO⁻) orbromamine species, and combinations thereof.

In further embodiments, the method for determining nitroreductase and/orhypoxia in a cell further comprises determining the presence of amembrane transporter MDR1, MRP or BCRP in the cell, by including atleast one xanthene compound that is transportable across a cell membraneby the membrane transporter in the incubation step (a), then determiningwhether the at least one xanthene compound is excluded from the cell,where the exclusion of the xanthene compound from the cell is indicativeof the presence of the membrane transporter. See, e.g., U.S. patentapplication Ser. No. 12/799,853, filed May 3, 2010. The presence of themembrane transporter in the cell may indicate that the cell is resistantto a chemotherapeutic agent. In some embodiments, this method furthercomprises comparing the exclusion of the xanthene compound from the cellwith the exclusion of the xanthene compound from a comparable celltreated with the xanthene compound and a membrane transporter inhibitor.

In still other embodiments, the method for determining nitroreductaseand/or hypoxia in a cell further comprises determining the location of asubcellular organelle in the cell, by including at least one dye thatlocalizes to the organelle in the incubation step (a), then visualizingthe location of the organelle by visualizing the at least one dye. See,e.g., U.S. Pat. Nos. 7,569,695 and 7,737,281 and U.S. PatentPublications US2009/0336954, 2010/0068752, 2010/0062460, and2010/0093004. As discussed in those publications, the organelle may be,e.g., the cell nucleus, the endoplasmic reticulum or the mitochondria.Additionally, the dye may be a cationic amphiphilic tracer compound thatlocalizes to a vacuole in a cell. An excess above normal accumulation ofvacuoles within the cell can be indicative of a lysosomal storagedisease.

As indicated in Example 6, hydrosulfite is a reducing agent that mimicsnitroreductases in that it is capable of reducing the nitro moiety onthe nitroreductase probes discussed above. Hydrosulfites, in particularsodium hydrosulfite, are sometimes used in food as a preservative.However, its use in foods is a concern due to its ability to cause acuteallergic reactions in sensitive individuals. It is therefore useful totest for hydrosulfites in foods. The nitroreductase probes can be usedfor that purpose.

Thus, the present invention is also directed to a method of detectinghydrosulfite in a sample, the method comprising

(a) combining the sample with any of the above-described compounds thatdetect nitroreductases, then

(b) determining whether SIG generates a greater signal than the signalgenerated by the compound when not exposed to a hydrosulfite. In theseembodiments, a greater signal indicates that the sample comprises ahydrosulfite.

In some embodiments of this method, the hydrosulfite is sodiumhydrosulfite.

The sample for these methods can be any sample suspected of containing ahydrosulfite. In some embodiments, the sample is a food.

Preferred embodiments are described in the following examples. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the examples.

Example 1. Synthesis of CD-1

a). Preparation of Intermediate CD-2

To a solution of 500 mg Rho110 in dimethylformamide, 290 μl ofN,N′-diisopropylethyl-amine was added and the mixture was stirred on icefor 10 minutes. Then 160 μl of N-morpholinecarbonyl chloride was addeddropwise. The reaction mixture was stirred on ice for an additional 15minutes and then at room temperature for 2 days. The reaction mixturewas evaporated to dryness and the oily residue was dissolved inchloroform containing a small amount of methanol. The product waspurified on a Biotage SP4 System with a SNAP 100 g column and achloroform methanol gradient.

b). Preparation of CD-1

To 23 mg of intermediate CD-2 dissolved in a mixture of dichloromethane(1 ml) and pyridine (1 ml), 40 mg of p-nitrobenzyl chloroformate inchloroform was added dropwise. The solution was stirred at roomtemperature for 2 days and the solvent was removed in vacuo. The resinwas co-evaporated with toluene, dissolved in chloroform and purified ona Biotage SP4 System with a SNAP 25 g column and a chloroform methanolgradient, to give 10 mg of a white product.

Example 2. Synthesis of CD-3

To 22 mg of CD-2 in the mixture of dichloromethane (0.7 ml), methanol(0.15 ml) and pyridine (1 ml), a solution of benzyl chloroformate (24mg) in chloroform was added dropwise. The reaction mixture was stirredat the room temperature for 3 days and the solvent was removed in vacuo.The resin was co-evaporated with toluene, dissolved in chloroform andpurified on a Biotage SP4 System with a SNAP 25 g column and achloroform methanol gradient, to give off-white crystals.

Example 3. Synthesis of CD-4

To 50 mg of Rho575 in dimethylformamide, 53 μl ofN,N′-diisopropylethylamine was added followed by 78 mg of p-nitrobenzylchloroformate dissolved in dimethylformamide. The reaction mixture wasstirred for 3 days and the solvent was removed in vacuo. The residue wasdissolved in chloroform and purified on a Biotage SP4 System with a SNAP25 g column and a chloroform methanol gradient, to give a pink product.

Example 4. Synthesis of CD-5

To 46 mg of 7-amino-4-trifluoromethylcoumarin in 250 μl ofdichloromethane, 300 μl of pyridine was added followed by 2.5equivalents of p-nitrobenzyl chloroformate dissolved in 250 μl ofdichloromethane. The reaction mixture was allowed to stir at roomtemperature overnight and another 2.5 equivalents of p-nitrobenzylchloroformate dissolved in dichloromethane were added. After 2 days thesolvent was removed in vacuo and the residue was coevaporated severaltimes with toluene. The resulting mixture was dissolved indichloromethane and purified on a Biotage SP4 System with a SNAP 25 gcolumn and a dichloromethane methanol gradient, to give an off-whiteproduct.

Example 5. Synthesis of CD-6

a). Preparation of CD-7

To a mixture of 55 mg 3-aminophenol, 95 mg 8-hydroxyjulolidine and 75 mgphthalic anhydride in 0.5 ml of DMF, a catalytic amount of anhydrouszinc chloride was added. The reaction mixture was irradiated in amicrowave oven in a closed vessel, at 100 W and 120° C. for 60 minutes,then evaporated to dryness. The solid residue was dissolved inchloroform containing a small amount of methanol. The product waspurified on the Biotage SP4 System with a SNAP 50 g column and achloroform methanol gradient.

b). Preparation of CD-6

To 30 mg of CD-7 in dimethylformamide, 32 μl ofN,N′-diisopropylethylamine was added followed by 47 mg of p-nitrobenzylchloroformate dissolved in dimethylformamide. The reaction mixture wasallowed to stir for 3 days and the solvent was removed in vacuo. Theresidue was dissolved in chloroform and purified on Biotage SP4 Systemwith SNAP 25 g column and chloroform methanol gradient to give off-whiteproduct.

Example 6. Nitroreductase Assays

(a) Chemical Assay

A solution of probe CD-6 in DMSO (0.1-1.0 μl) was added to 90 μl of PBSbuffer (10 mM, pH 7.4). After mixing, 10 μl of 100 mM aqueous sodiumhydrosulfite was added. The reaction mixture was incubated at roomtemperature for amount of time ranging from 1 minute to 6 hours. As acontrol, 10 μl of water instead of sodium hydrosulfite was used. FIG. 2illustrates increasing fluorescence of CD-6 over a 60 minute periodafter combining with sodium hydrosulfite.

(b) Enzymatic Assay

E. coli nitroreductase (Sigma-Aldrich, St. Louis, Mo.) at 10 mg/ml wasincubated in PBS buffer (pH 7.4) with a DMSO solution of a given hypoxiamarker in the presence of 500 μM NADH (Sigma-Aldrich, St. Louis, Mo.).The incubation was carried out at 37° C. for 1 h. Small samples of thereaction mixture were taken for spectral analysis at several time points(typically at 5, 15, 30 and 60 min).

Example 7. Detection of Hypoxia with Hypoxia Markers Using FluorescenceMicroscopy

The human cervical adenocarcinoma epithelial cell line HeLa, U-2 OShuman bone osteosarcoma cell line and hamster ovary CHO K1 cell linewere obtained from ATTC. HeLa cells were routinely cultured in Eagle'sMinimum Essential Medium with low glucose (ATCC), supplemented with 10%fetal bovine serum (ATCC) and 100 U/ml penicillin, 100 μg/mlstreptomycin (Sigma-Aldrich, St. Louis, Mo.). U-2 OS cells wereroutinely cultured in McCoy's 5a Modified Medium (ATCC), supplementedwith 10% fetal bovine serum heat (ATCC) and 100 U/ml penicillin+100μg/ml streptomycin (Sigma-Aldrich). CHO K1 cells were cultured in F-12Kmedium (ATCC), supplemented with 10% fetal bovine serum (ATCC) and 100U/ml penicillin+100 μg/ml streptomycin (Sigma-Aldrich). Cell cultureswere maintained in an incubator at 37° C., in a 5% CO₂ atmosphere.

The following stocks of hypoxia inducers were prepared: 200 mM of CoCl₂in water (1000×), 50 mM of DFO (desferrioxamine, 250×) in DMSO or 250 mMof DMOG (dimethyloxalylglycine, N-[Methoxyoxoacetyl]glycine methylester, 250×) in DMSO. Stock solutions of the inducers were aliquoted andstored at −20° C. CD-1 and the control compound CD-3 were synthesized asdescribed in Examples 1 and 2. Stock solutions (10 μM in DMSO, 2000×) ofthe probe and the control substances were prepared, aliquoted and storedat −20° C. in the dark. HP-1 kit for hypoxia detection (Hypoxyprobe Inc,Burlington, Mass.) was employed to confirm hypoxia induction in thecells.

The day before the experiment, the cells were seeded on 4-wellmicroscope slides (Cel-Line™ Brand, Portsmouth, N.H.) at a density 1×10⁴cells/well (2×10⁴ cells/cm²). On the day of the experiment, the cellswere pre-loaded with either the hypoxia probe or control (5 μM finalconcentration in cell culture medium), and a hypoxic state was inducedin the cells by the treatment with the following hypoxia inducers for3.5 h at 37° C. and 5% CO₂: 0.2 mM of CoCl₂, 0.2 mM of DFO or 1 mM ofDMOG. Alternatively, hypoxia was induced by the incubation of the cellsfor 3.5 h at 37° C. in a Billup-Rothenberg chamber (Billup-Rothenberg,Inc., San Diego, Calif.) in an anoxic environment (95% N₂, 5% CO₂).Post-treatment, the slides were washed twice with PBS, coverslipped andvisualized using an Olympus BX-51 fluorescence microscope (FITC filterset, 490ex/525em).

In parallel, the hypoxia cellular state was detected using the hypoxiadetecting reagent pimonidazole (Varia et al., 1998; Young, 1977).Briefly, the cells on the slides were preloaded with 0.2 mM pimonidazolein culture medium, treated with hypoxia inducers as described above,washed twice with PBS, fixed in methanol for 10 min at −20° C.,re-hydrated in PBS, then blocked overnight at 4° C. using 3% BSA in PBS.Slides were stained with a 1:100 dilution of FITC-MAb1 againstpimonidazole (HP-1 kit from Hypoxyprobe) for 45 min at 4° C., washedtwice with PBS, coverslipped and visualized using an Olympus BX-51fluorescence microscope equipped with an FITC filter set (490ex/525em).Cells not treated, not loaded with pimonidazole and not stained withFITC-MAb1 were used as negative controls.

The results of staining hypoxic HeLa cells are presented in FIG. 3. CD-1was reduced by the nitroreductase enzyme present in hypoxic cells (bothpost-chemical induction of hypoxia, FIG. 3A, and anoxic treatment, lefthalf of FIG. 3C) and the resulting reduction product spontaneouslydecomposed yielding a bright fluorescence signal. The control, CD-3,that cannot be reduced by nitroreductase did not yield any fluorescentsignal (FIG. 3B and right half of FIG. 3C). The fluorescence patternobserved in hypoxic cells stained with CD-1 correlated with the patternobtained after staining the cells with pimonidazole and fluorescentlylabeled monoclonal antibody (FIG. 3D). Staining of U-2 OS and CHO K1cells treated with cobalt chloride, DFO or DMOG or subjected to anoxiademonstrated similar results (data not shown).

Example 8. Validation of Multi-Color Hypoxia Markers CD-1, CD-4, CD-5and CD-6 Using Fluorescent Microscopy

HeLa and U-2 OS cells were cultured as described in Example 7. CompoundsCD-1, CD-4, CD-5 and CD-6 were dissolved in anhydrous DMSO at 10 mMconcentration (1000× stock solutions). Stock solutions of the dyes werealiquoted and stored at −20° C. in the dark. All hypoxia inducer stockswere prepared as described in Example 7.

The day before the experiment, the cells were seeded on 4-wellmicroscope slides (Cel-Line™ Brand, Portsmouth, N.H.) at a density 1×10⁴cells/well (2×10⁴ cells/cm²). On the day of the experiment, the cellswere preloaded with the hypoxia probes (5 μM final concentration forCD-1 and CD-5 probes, 1 μM final concentration for CD-4 and CD-6 probesin cell culture medium), and hypoxia was induced in the cells by thetreatment with the following hypoxia inducers for 3.5 h at 37° C. and 5%CO₂: 0.2 mM of CoCl₂, 0.2 mM of DFO or 1 mM of DMOG. Alternatively,hypoxia was induced by the incubation of the cells for 3.5 h at 37° C.in a Billup-Rothenberg chamber (Billup-Rothenberg, Inc.) in an anoxicenvironment (95% of N₂, 5% of CO₂). Post-treatment, the slides werewashed twice with PBS, coverslipped and visualized using an OlympusBX-51 fluorescence microscope with a DAPI filter set (350 ex/470em) forCD-5, an FITC filter set (490ex/525em) for CD-1, an orange filter set(550ex/620em) for CD-4 and a Texas Red filter set (596ex/670em) forCD-6.

Each tested self-immolative probe got efficiently processed in hypoxicHeLa cells yielding bright fluorescence signal in the corresponding areaof spectrum (FIGS. 4A-4D). Untreated cells did not show anyfluorescence.

Example 9. Detection of Hypoxia with CD-1, CD-4 and CD-6 Using FlowCytometry

HeLa and U-2 OS cells were cultured as described in Example 7. HumanJurkat T-cell leukemia cells (the A3 subclone) was obtained from ATCCand routinely cultured in RPMI-1640 medium (ATCC) supplemented with 10%fetal bovine serum (ATCC) and 100 U/ml penicillin+100 μg/ml streptomycin(Sigma-Aldrich). Cell cultures were maintained in an incubator at 37°C., with 5% CO₂ atmosphere. CD-1, CD-4 and CD-6 self-immolative hypoxiaprobes were prepared as described in Example 8. All hypoxia inducersstocks were prepared as described in Example 7.

The day before the experiment, HeLa and U-2 OS cells were seeded in6-well tissue culture plates at a density 5×10⁵ cells/well. Jurkat cellswere collected in logarithmic phase of growth and aliquoted at a density5×10⁵ cells/sample. On the day of the experiment, the cells werepreloaded with the hypoxia probes (5 μM final concentration for CD-1, 1μM final concentration for CD-4 and CD-6 in cell culture medium), andthe hypoxic state was induced in the cells as described in the Example7. Post-treatment, the adherent cells (HeLa and U-2 OS) were collectedby trypsinization, re-suspended in 0.5 mL of fresh PBS and analyzedusing flow cytometry. Jurkat cells were analyzed without washing.

Flow cytometry experiments were performed using a FACS Calibur benchtopflow cytometer (BD Biosciences) equipped with a blue (488 nm) laser, andthe fluorescence was recorded in the FITC (530/30 filter), PE (585/42filter) and PerCP (670 LP filter) channels. Hypoxic cells were detectedby detecting increases in fluorescence. The degree of hypoxia (K_(hyp))can be quantified by using Kolmogorov-Smirnov statistics (Young, 1977)or the following formula:K _(hyp)=(MFI_(hyp)−MFI_(con))/MFI_(hyp),where MFI_(hyp) and MFI_(con) are median fluorescent intensities ofhypoxia-induced and control cells, respectively. Alternatively,quantification can be approached by using quadrant and/or regionsstatistics that are usually embedded in flow cytometry software. Thedata, shown in FIG. 5, demonstrates a fluorescence increase in Jurkatcells loaded with probes CD-1 (panel A) or CD-6 (panel B) and treatedwith chemical hypoxia inducers (CoCl₂, DFO, DMOG) or subjected toanoxia. Cells loaded with the corresponding probes and treated withvehicle only are considered to be controls. The numbers on thehistograms indicated the degree of hypoxia determined using the formulaabove (the values over 20 indicate hypoxic cellular state). Similarresults were obtained for HeLa and U-2 OS cells (data not shown).

Example 10. Combined Detection of Hypoxia and Redox Status in Live Cellsby Fluorescent Microscopy Using CD-1 and CD-5 Hypoxia Probes andReactive Oxygen Species Detection Reagents

HeLa and U-2 OS cells were cultured as described in Example 7. Stocksolutions of CD-1 and CD-5 and various hypoxia inducers were preparedand stored as described in Examples 7 and 8. Additionally, 5 mM stocksolutions (5000×) of 2′,7′-dichloro-fluorescein diacetate (DCFDA, anindicator of global ROS generation) and dihydroethidium (DHE, specificindicator of superoxide generation) were made in anhydrous DMF. DMSO wasavoided, since this solvent is a hydroxyl radical scavenger and itspresence may affect ROS/RNS production in cellular systems. Thefollowing stocks of ROS inducers were prepared in DMF: 10 mM pyocyanin(general ROS generating compound, 100×), 20 mM antimycin A (general ROSinducer, 400×), 50 mM pyrogallol (a superoxide radical generator,1000×), 1 mM t-butyl hydroperoxide (TBHP, peroxide radical inducer,10,000×). ROS probes and inducers were aliquoted and stored at −80° C.

The day before the experiment, the cells were seeded on 4-wellmicroscope slides (Cel-Line™ Brand, Portsmouth, N.H.) at a density 1×10⁴cells/well (2×10⁴ cells/cm²). On the day of the experiment, the cellswere preloaded with both hypoxia probe (5 μM final concentration in cellculture medium) and ROS detecting reagent with suitable spectralcharacteristics. Two ROS-detecting probes, DCFDA and DHE, were used in apilot experiment in conjunction with hypoxia self-immolative probes. Thehydrolyzed product of DCFDA, DCFH, is considered to be a generalindicator of ROS, reacting with H₂O₂ (in the presence of peroxidases),ONOO⁻, lipid hydroperoxides, and hydroxyl radicals. The oxidized productcan be detected by strong fluorescence emission at around 525 nm whenexcited at around 488 nm. Because H₂O₂ is a secondary product of O₂^(•−), DCFH fluorescence has been used to implicate O₂ ^(•−) production.The direct reaction of DHE with O₂ ^(•−) yields a very specificfluorescent product, requiring no conversion to H₂O₂. The product of theDHE reaction with O₂ ^(•−) fluoresces strongly at around 600 nm whenexcited at 500-530 nm.

Tested pairs of the probes were as follows: CD-5 with DCFDA or DHE, andCD-1 with DHE. A hypoxic state was induced in the cells as described inExample 7. Control slides were singly-stained with hypoxia orROS-detecting probes only. Additional slides were treated with differentgeneral and specific ROS inducers and stained with ROS-detecting probesto control ROS staining. Post-treatment, the slides were washed twicewith PBS, coverslipped and visualized using an Olympus BX-51fluorescence microscope and a DAPI filter set (350 ex/470em) for CD-5,an FITC filter set (490ex/525em) for CD-1 or for total ROS detectionreagent, and an orange filter set (550ex/620em) for HE.

The results of this experiment are presented in FIGS. 6A-6C. While CD-1and CD-5 probes generated bright fluorescent signal in all four hypoxicsamples of HeLa cells (anoxia-exposed or treated with CoCl₂, DFO andDMOG), ROS-detecting dyes give more individually distinct results.Abundant superoxide production was detected in anoxic DFO andDMOG-treated cells, while in CoCl₂-treated samples there was onlymoderate staining with the superoxide detection reagent. At the sametime, peroxides/peroxynitrite formation was detected in DMOG-treatedcells only. Comparison of the result of the double staining with hypoxiaand ROS detecting probes with the results of separate staining witheither probe validated obtained results and demonstrated thatabove-mentioned probes are compatible for multiplexed detection of bothhypoxia and/or redox status of the cell.

Example 11. Combined Detection of Hypoxia and Superoxide in Live Cellsby Flow Cytometry Using CD-1 and CD-6 Hypoxia Probes and SuperoxideDetection Reagent

HeLa, U-2 OS, and Jurkat cell lines were cultured as described inExamples 7 and 9. Stock solutions of the probes and inhibitors wereprepared as described in Examples 7 and 10.

The day before the experiment, HeLa and U-2 OS cells were seeded in6-well tissue culture plates at a density of 5×10⁵ cells/well. Jurkatcells were collected in logarithmic phase of growth and aliquoted at adensity of 5×10⁵ cells/sample. On the day of the experiment, the cellswere preloaded with either hypoxia or ROS-detecting probes only or withthe combination of hypoxia and ROS-detecting probes with compatiblespectral characteristics. Samples stained with single hypoxia probes orwith two ROS-detecting probes were made to validate data obtained withthe combination of hypoxia and redox probes. The probes used in theexperiment and their final concentrations in cell culture medium were asfollows: CD-1 (5 μM) and CD-6 (1 μM), DCFDA,peroxide/peroxynitrite/hydroxyl detection probe and HE, specificsuperoxide detection reagent (1 μM for both redox probes). Hypoxia wasinduced in the cells as described in Example 7. Additionally, cells weretreated with different general and specific ROS inducers and stainedwith ROS-detecting probes to control ROS production and staining.Post-treatment, the adherent cells (HeLa and U-2 OS) were collected bytrypsinization, re-suspended in 0.5 mL of fresh PBS and analyzed usingflow cytometry. Jurkat cells were analyzed without washing. Flowcytometry experiments were performed using an FACS Calibur benchtop flowcytometer (BD Biosciences) equipped with a blue (488 nm) laser, and thefluorescence was recorded in the FITC (530/30 filter), PE (585/42filter) and PerCP (670 LP filter) channels. Data were collecteduncompensated, also compensation corrections were performed usingunstained cells, and cells stained with each single dye separately(CD-1, CD-6, DCFDA or HE). To quantify data, quadrant gates were setusing untreated samples.

The results of pilot experiments are presented in FIGS. 7 and 8. WhenCD-1 was used in combination with the superoxide detection reagent inJurkat cells treated as described earlier, the increased population ofgreen positive cells was detected by flow cytometry in eachhypoxia-induced sample (FIG. 7A). Simultaneously, significant superoxideproduction was detected in anoxic, DFO- and DMOG-treated samples byincreased population (40% and more compared to the untreated cells) oforange positive cells. In CoCl₂-treated samples, superoxide productionwas moderate—about 20% of orange positive cells. Pyocyanin-treated cells(positive control for ROS production) display high superoxide production(as expected) and a moderate hypoxia state (about 10% of the positivegreen cells). Cells stained with the CD-1 probe only exhibit similarnumbers for green positive populations (FIG. 7B). Staining withROS-detecting reagents also corroborated superoxide production data forhypoxia-induced Jurkat cells (FIG. 7C).

Similar results were obtained for the combination of CD-6 with DCFDA orwith HE dyes (FIG. 8). The hypoxia probe was able to detect hypoxic cellpopulations when employed in combination with any of ROS detecting dyes(FIGS. 8A and 8B) or alone (FIG. 8C). The data was corroborated bystaining of pyocyanin-treated cells (positive control for ROSproduction) and also by analyzing samples stained with ROS-detectingdyes (FIG. 8D).

Example 12. Detection of Hypoxia in Live Cells Using Hypoxia Probes andMultiplate Fluorescence Reader

HeLa, U-2 OS, and Jurkat cell lines were cultured as described inExamples 7 and 9. Stock solutions of the probes and inducers wereprepared as described in Examples 7 and 8. Two protocols (using cells insuspension and using adherent cells) were used.

A. For the first protocol (suspension cells), cells in suspension (5×10⁵cells/100 μL) were added to the wells of 96-well black walledmicroplates where medium containing hypoxia probes (5 μM finalconcentration for CD-1 and CD-5, 1 μM for CD-4 and CD-6) and chemicalhypoxia inducers (CoCl₂, DFO or DMOG) or vehicle were added.Additionally hypoxia was induced by incubation of the cells in anoxicenvironment (Billup-Rothenberg chamber, 5% CO₂, 95% N₂) as described inExample 6. Cells were incubated for 3.5 hrs at 37° C. Fluorescence wasthen immediately measured by fluorescence microplate reader.Alternatively, after the incubation, plates were centrifuged (5 min,200×g), the supernatant was removed, the cells were resuspended in 200μL of cold tissue culture medium and retained fluorescence was measured.In some cases, cells were washed with cold PBS after staining.

B. For adherent cells, the cells were seeded in black walled 96-wellmicroplates (0.5-2.0×10⁵ cells/well). Twenty four hrs later, mediumcontaining hypoxia probes (5 μM final concentration for CD-1 and CD-5, 1μM for CD-4 and CD-6) and chemical hypoxia inducers (CoCl₂, DFO or DMOG)or vehicle were added to the cells for 3.5 hrs at 37° C. Additionallyhypoxia was induced by incubation of the cells in an anoxic environment(Billup-Rothenberg chamber, 5% CO₂, 95% N₂). Fluorescence was thenmeasured by fluorescence microplate reader immediately or after excessdye(s) was removed by washing with PBS.

The probe(s) fluorescence was measured using an OPTIMA FluoStarmultiplate fluorimeter (BMG Labtech Inc., N.C.) equipped with 340, 490and 550 nm excitation filter and 480, 520, 570 and 610 nm emissionfilters, or Synergy™ Mx BioTek multi-mode microplate reader (BioTekInstruments Inc., Vt.) using 378, 490 and 520 nm excitation and 488,520, 540 and 600 nm emission settings. The results of the assay werenormalized to the fluorescence of the empty well and expressed as aratio of the fluorescence of the inducer-treated cells to thefluorescence of the untreated control cells.

Results of hypoxia detection using a fluorescence microplate reader arepresented in FIG. 9 and are similar to the results obtained byfluorescence microscopy and flow cytometry methods.

Example 13. Synthesis of CD-8

a). Preparation of CD-9

To 100 mg of 4-amino-N-methylphthalimide in 3 ml of dimethylformamide,0.5 ml of pyridine was added followed by 300 mg of p-nitrobenzylchloroformate. The reaction mixture was allowed to stir overnight andthe solvent was removed in vacuo. The residue was dissolved inchloroform and purified on Biotage SP4 System with SNAP 25 g column andchloroform methanol gradient to give off-white product (110 mg).

b). Preparation of CD-8

To 20 mg of CD-9 in 3 ml of methanol, 0.5 ml of hydrazine was added. Thereaction mixture was stirred for 1 hour at room temperature and thesolvent was removed in vacuo. The residue was dissolved in chloroformand purified on Biotage SP4 System with SNAP 25 g column and chloroformmethanol gradient to give off-white product (15 mg).

Example 14. Chemical Reduction of CD-8 and Enzymatic Testing of theReduction Product

100 μl of 20004 CD-8 (dissolved in 1×PBS buffer) with (1 mM) or withoutsodium hydrosulfite was incubated in a 96 black microplate (Greiner®) atroom temperature for 1 hour. Also included was a positive control of 100μl of 50 μM of isoluminol dissolved in 1×PBS buffer. A 4:1 volumetricmixture (1.25 μL) of stabilized H₂O₂ (Roche Diagnotics GmbH: Referencenumber: 11 582 950 001, component 2) and 200 mM of 4-idophenol was thenadded. Before adding horseradish peroxidase (HRP), the backgroundluminescence was recorded; after adding 2 μL of 50 μM HRP, theluminescence was immediately recorded on a Biotek® plate reader. Theresults are summarized in FIG. 10. Sodium hydrosulfite clearly triggeredthe reduction of the nitro group of CD-8 into an amino group, triggeringthe self-immolative release of isoluminol.

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In view of the above, it will be seen that several objectives of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

What is claimed is:
 1. A compound for detecting histone deacetylaseactivity, said compound consisting of the structure:

wherein SIG is a signaling moiety, wherein SI is a self-immolativestructure bound to SIG such that SIG has a reduced signal relative tothe signal of SIG without SI, wherein m is an integer from 1 to 10, andwherein, when the compound is modified by histone deacetylase activity,SI is destabilized and self-cleaved from SIG such that SIG generates anincreased signal.
 2. The compound of claim 1, wherein SIG is afluorescent moiety or a luminescent moiety.
 3. The compound of claim 1,wherein m is an integer from 2 to
 10. 4. The compound of claim 3,wherein SIG is a fluorophore selected from a symmetric or asymmetriccyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, acoumarin, an iminocoumarin, a xanthene, a rhodamine, a rhodaminederivative, a rosamine, a rosamine derivative, a rhodol or a rhodolderivative.
 5. The compound of claim 1, wherein m=1.
 6. The compound ofclaim 5, wherein SIG is a fluorophore selected from a symmetric orasymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, axanthene, a coumarin, an iminocoumarin, a xanthene, a rhodamine, arhodamine derivative, a rosamine, a rosamine derivative, a rhodol or arhodol derivative.
 7. The compound of claim 1, consisting of thestructure:

wherein each R¹ is independently a hydrogen, a halogen, Z, a cyanogroup, an isocyano group, a thiocyano group, an isothiocyano group, anazido group, a trihalomethyl group; a sulfonate group, a sulfate group,a carboxyl group, a carbonyl group, an ester group, an amido group, acarbamate group, a phosphate group, a phosphonate group, an amino group,an alkoxy group, a thiol group, a sulfoxy group, a sulfone group, asulfonamide group, a phosphino group, or a silane group, wherein Z is areducible nitrogen-containing group, or an amino group bound to anelectron deficient moiety, and wherein n is 0, 1, 2, 3, or
 4. 8. Thecompound of claim 7, wherein SIG is a fluorescent moiety or aluminescent moiety.
 9. The compound of claim 7, wherein m is an integerfrom 2 to
 10. 10. The compound of claim 9, wherein SIG is a fluorophoreselected from a symmetric or asymmetric cyanine, a merocyanine, a styrylmoiety, an oxazine, a xanthene, a coumarin, an iminocoumarin, axanthene, a rhodamine, a rhodamine derivative, a rosamine, a rosaminederivative, a rhodol or a rhodol derivative.
 11. The compound of claim7, wherein m=1.
 12. The compound of claim 11, wherein SIG is afluorophore selected from a symmetric or asymmetric cyanine, amerocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin, animinocoumarin, a xanthene, a rhodamine, a rhodamine derivative, arosamine, a rosamine derivative, a rhodol or a rhodol derivative.
 13. Amethod for detecting histone deacetylase activity in a sample, themethod comprising (a) incubating the sample with the compound of claim 1for a time and under conditions sufficient for the compound to bemodified by a histone deacetylase if present in the sample; and (b)measuring the signal generated by SIG, wherein the amount of signalgenerated is proportional to the amount of histone deacetylase activityin the sample.
 14. The method of claim 13, wherein the sample is a fluidof an organism or a colony of organisms, or an extract thereof.
 15. Amethod for detecting histone deacetylase activity in a sample, themethod comprising (a) incubating the sample with the compound of claim 7for a time and under conditions sufficient for the compound to bemodified by a histone deacetylase if present in the sample; and (b)measuring the signal generated by SIG, wherein the amount of signalgenerated is proportional to the amount of histone deacetylase activityin the sample.
 16. The method of claim 15, wherein the sample is a fluidof an organism or a colony of organisms, or an extract thereof.
 17. Acompound consisting of the structure:


18. The compound of claim 17, consisting of the structure:


19. The compound of claim 17, consisting of the structure:


20. The compound of claim 17, consisting of the structure:


21. A method for detecting histone deacetylase activity in a sample, themethod comprising (a) incubating the sample with the compound of claim17 for a time and under conditions sufficient for the compound to bemodified by a histone deacetylase if present in the sample; and (b)measuring the luminescent signal generated by the compound, wherein theamount of signal generated is proportional to the amount of histonedeacetylase activity in the sample.
 22. The method of claim 21, whereinthe sample is a fluid of an organism or a colony of organisms, or anextract thereof.